The present invention relates generally to the optical characterization of biological particles using a flow cytometer. More particularly, the present invention teaches a method and apparatus for the sizing of individual biological particles in a flow cytometer using measured forward angle light scatter and for obtaining a particle fluorescence intensity measurement that is surface area or volume normalized.
It will be appreciated by those skilled in the art that the use of flow cytometric methods in the study of cell populations, surface receptors, and DNA content has greatly enhanced the understanding of many disease states. Patterns in forward and right angle light scatter are routinely used to select subpopulations of cells for further characterization of cell surface and cytoplasmic antigens by fluorescently conjugated probes. Typically, flow cytometric results are reported as the mean fluorescence intensity or mean fluorescence channel of the population being measured. Most published studies equate changes in the fluorescence intensity of a specific cell population with alterations in biological activity.
Fluorescence intensity measurements are unambiguous only if the physical properties of each event are constant. Prior art measurement methods do not take into account the fact that fluorescence intensity measurements are directly related to the size of the particle measured as well as to the surface distribution of the fluorescent probe. Generally, prior art flow cytometry particle characterization methods have not recognized the need to adjust the measured fluorescence for deviations in cell size, whether caused by cell cycle, size abnormalities, activation, aggregation, or other factors.
Much theoretical and practical experimentation was conducted during the 1970's concerning optical characterization of biological cells by classical light scatter techniques. There are three main points which are apparent from review of these studies. First, all studies agree that sizing of biological cells is possible using forward angle light scatter measurements. These studies report accurate sizing in both static and flow systems. Second, the presence of internal structures significantly alters light scatter measured at large scattering angles, but has little to no affect on the angular dependence of light scatter at angles below 10.degree.. Third, the contribution of refracted light is small and only a weak function of size in the forward direction. Fraunhofer diffraction theory has been used as the basis of successful cell sizing.
A particle's size, shape, texture, and internal structures may be characterized by analysis of the intensity and angles through which light is scattered. Much theoretical work has been accomplished describing the mechanism and results of light scatter by small particles. Equations have been developed which relate light scatter to the illumination wavelength, refractive index, angle of scatter, and particle size. Mie theory, Fraunhofer diffraction theory, photon correlation spectroscopy, turbidity spectra, and Rayleigh Debye regimes can accurately predict light scattered from small particles within a specified range of conditions.
Light scatter can be divided into three distinct categories: diffraction, refraction, and reflection. Forward angle light scatter (FALS), as measured in a flow cytometer, is composed primarily of diffracted light, while right angle light scatter (RALS) is composed of significant proportions of all light scatter components. Diffracted light collected within small forward angles is rich in particle size information. Since very little refracted light is collected in the FALS detector, internal structures do not influence the FALS measurement unless the particle and its internal structures are very large and well defined. Particle shape, orientation, and degree of well defined internal structures influence refracted and reflected light scatter primarily through large angles, as can be seen in RALS flow cytometer measurements. The use of RALS to distinguish cell populations based on internal structures has been successfully described in the past.
While FALS is generally considered to be a rough measure of cell size, the full capacity of forward angle light scatter in flow cytometry has not been realized. Implementation of a theoretical model for diffracted light provides valuable particle size information from the flow cytometer. Fraunhofer diffraction theory (FDT) accurately predicts diffracted light scatter in the particle size range from 0.1 to 700 microns in diameter, depending on the illuminating wavelength and the angle of collection. FDT is of particular value for cellular studies using flow cytometers due to its simplicity and the cell-size particle range it describes.
Flow cytometry allows detection of molecular level changes which occur on the surface of individual biological cells based on the binding of fluorescent probes. Interpretation of flow cytometric data describing such changes, such as would occur in platelet activation, is complicated since the fluorescence intensity measurements are directly related to the particle size, as well as the surface distribution of bound fluorescent probes. Prior art optical techniques allow an estimate of the particle size distribution, but do not provide individual particle sizes. Resistive techniques do provide individual particle sizes, but do not have the capability to measure single particle fluorescence. Size complications necessitate the development of a new quantitative flow cytometric technique which decouples particle fluorescence and particle size.
Thus, there is a need for a method of accurately determining particle radius from forward angle light scatter measured in a flow cytometer. Further, a quantitative flow cytometric method based on surface area or volume normalization is lacking in the prior art. Such a method, uniquely provided by optical sizing, would allow quantitation of fluorescent probe binding independent of particle size.